Roger Huerlimann

Growth, Lipid Content, Productivity, and Fatty Acid Composition of Tropical Microalgae for Scale-Up Production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m−2 day−1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m−2 day−1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257.

Comprehensive guide to acetyl-carboxylases in algae

Lipids from microalgae have become an important commodity in the last 20 years, biodiesel and supplementing human diets with ω-3 fatty acids are just two of the many applications. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the lipid synthesis pathway. In general, ACCases consist of four functional domains: the biotin carboxylase (BC), the biotin carboxyl binding protein (BCCP), and α-and ß-carboxyltransferases (α-and ß-CT). In algae, like in plants, lipid synthesis is another function of the chloroplast. Despite being well researched in plants and animals, there is a distinct lack of information about this enzyme in the taxonomically diverse algae. In plastid-containing organisms, ACCases are present in the cytosol and the plastid (chloroplasts) and two different forms exist, the heteromeric (prokaryotic) and homomeric (eukaryotic) form. Despite recognition of the existence of the two ACCase forms, generalized published statements still list the heteromeric form as the one present in algal plastids. In this study, the authors show this is not the case for all algae. The presence of heteromeric or homomeric ACCase is dependent on the origin of plastid. The authors used ACCase amino acid sequence comparisons to show that green (Chlorophyta) and red (Rhodophyta) algae, with the exception of the green algal class Prasinophyceae, contain heteromeric ACCase in their plastids, which are of primary symbiotic origin and surrounded by two envelope membranes. In contrast, algal plastids surrounded by three to four membranes were derived through secondary endosymbiosis (Heterokontophyta and Haptophyta), as well as apicoplast containing Apicomplexa, contain homomeric ACCase in their plastids. Distinctive differences in the substrate binding regions of heteromeric and homomeric α-CT and β-CT were discovered, which can be used to distinguish between the two ACCase types. Furthermore, the acetyl-CoA binding region of homomeric α-CT can be used to distinguish between cytosolic and plastidial ACCase. The information provided here will be of fundamental importance in ACCase expression and activity research to unravel impacts of environmental and physicochemical parameters on lipid content and productivity.

The effect of nitrogen limitation on acetyl-CoA carboxylase expression and fatty acid content in Chromera velia and Isochrysis aff. galbana (TISO).

Lipids from microalgae have become a valuable product with applications ranging from biofuels to human nutrition. While changes in fatty acid (FA) content and composition under nitrogen limitation are well documented, the involved molecular mechanisms are poorly understood. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the FA synthesis and elongation pathway. Plastidial and cytosolic ACCases provide malonyl-CoA for de novo FA synthesis in the plastid and FA elongation in the endoplasmic reticulum, respectively. The present study aimed at investigating the expression of plastidial and cytosolic ACCase in Chromera velia and Isochrysis aff. galbana (TISO) and their impact on FA content and elongation level when grown under nitrogen-deplete conditions. In C. velia, plastidial ACCase was significantly upregulated during nitrogen starvation and with culture age, strongly correlating with increased FA content. Conversely, plastidial ACCase of I. aff. galbana was not differentially expressed in nitrogen-deplete cultures, but upregulated during the logarithmic phase of nitrogen-replete cultures. In contrast to plastidial ACCase, the cytosolic ACCase of C. velia was downregulated with culture age and nitrogen-starvation, strongly correlating with an increase in medium-chain FAs. In conclusion, the expression of plastidial and cytosolic ACCase changed with growth phase and nutrient status in a species-specific manner and nitrogen limitation did not always result in FA accumulation.

Toxic effects of polyethylene terephthalate microparticles and Di(2-ethylhexyl)phthalate on the calanoid copepod, Parvocalanus crassirostris

Large amounts of plastic end up in the oceans every year where they fragment into microplastics over time. During this process, microplastics and their associated plasticizers become available for ingestion by different organisms. This study assessed the effects of microplastics (Polyethylene terephthalate; PET) and one plasticizer (Di(2-ethylhexyl)phthalate; DEHP) on mortality, productivity, population sizes and gene expression of the calanoid copepod Parvocalanus crassirostris. Copepods were exposed to DEHP for 48h to assess toxicity. Adults were very healthy following chemical exposure (up to 5120µg L-1), whereas nauplii were severely affected at very low concentrations (48h LC50value of 1.04 ng L-1). Adults exposed to sub-lethal concentrations of DEHP (0.1-0.3µg L-1) or microplastics (10,000-80,000 particles mL-1) exhibited substantial reductions in egg production. Populations were exposed to either microplastics or DEHP for 6 days with 18 days of recovery or for 24 days. Populations exposed to microplastics for 24 days significantly depleted in population size (60±4.1%, p<0.001) relative to controls, whilst populations exposed for only 6 days (with 18 days of recovery) experienced less severe depletions (75±6.0% of control, p<0.05). Populations exposed to DEHP, however, exhibited no recovery and both treatments (6 and 24 days) yielded the same average population size at the termination of the experiment (59±4.9% and 59±3.4% compared to control; p<0.001). These results suggest that DEHP may induce reproductive disorders that can be inherited by subsequent generations. Histone 3 (H3) was significantly (p<0.05) upregulated in both plastic and DEHP treatments after 6 days of exposure, but not after 18 days of recovery. Hsp70-like expression showed to be unresponsive to either DEHP or microplastic exposure. Clearly, microplastics and plasticizers pose a serious threat to zooplankton and potentially to higher trophic levels.

Microalgal classification: major classes and genera of commercial microalgal species

Microalgae show enormous promise to simultaneously create industries aiming for remediation of wastewaters and producing value-adding coproducts for large nonsaturable markets, such as biofuels, biofertilizers, and biopolymers. To date, however, their commercially realized exploitation is restricted to a few strains for the production of fine chemicals, nutraceuticals, food additives, and as feed in aquaculture. Algal taxonomy has remained a mystery for applied phycologists. In this chapter, we aim to unriddle this puzzle by providing a brief overview of the difficulties of placing microalgae in a hierarchical, phylogenetically meaningful, classification scheme. The evolutionary origin of algal diversity is discussed, which explains why creating a phylogenetically meaningful classification scheme is fraught with difficulties. Finally, commercially used microalgae or those with demonstrated potential are described with a focus on their identifying characteristics. These described microalgae are then cross-linked to their remediation and bioproduct potential in the last two sections of this chapter.

Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO)

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω-3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ∆(6)-, ∆(8)-, ∆(5)- and ∆(4)-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.

Response of mixed methanotrophic consortia to different methane to oxygen ratios

Methane (CH4) and oxygen (air) concentrations affect the CH4 oxidation capacity (MOC) and mixed methanotrophic community structures in compost (fresh) and landfill (age old) top cover soils. A change in the mixed methanotrophic community structure in response has implications for landfill CH4 bio-filter remediation and possible bio-product outcomes (i.e., fatty acid methyl esters (FAME) content and profiles and polyhydroxybutyrate (PHB) contents). Therefore the study aimed to evaluate the effect of variable CH4 to oxygen ratios (10-50% CH4 in air) on mixed methanotrophic community structures enriched from landfill top cover (LB) and compost soils (CB) and to quantify flow on impacts on MOC, total FAME contents and profiles, and PHB accumulation. A stable consortium developed achieving average MOCs of 3.0±0.12, 4.1±0.26, 6.9±0.7, 7.6±1.3 and 9.2±1.2mgCH4g-1DWbiomassh-1 in LB and 2.9±0.04, 5.05±0.32, 6.7±0.31, 7.9±0.61 and 8.6±0.48mgCH4g-1DWbiomassh-1 in CB for a 20day cultivation period at 10, 20, 30, 40 and 50% CH4, respectively. CB at 10% CH4 had a maximal FAME content of 40.5±0.8mgFAMEg-1DWbiomass, while maximal PHB contents (25mgg-1DWbiomass) was observed at 40% CH4 in LB. Despite variable CH4/O2 ratios, the mixed methanotrophic community structures in both LB and CB were relatively stable, dominated by Methylosarcina sp., and Chryseobacterium, suggesting that a resilient consortium had formed which can now be tested in bio-filter operations for CH4 mitigations in landfills.

Fecal bacterial communities of wild-captured and stranded green turtles (Chelonia mydas) on the Great Barrier Reef

ABSTRACT Green turtles (Chelonia mydas) are endangered marine herbivores that break down food particles, primarily sea grasses, through microbial fermentation. However, the microbial community and its role in health and disease is still largely unexplored. In this study, we investigated and compared the fecal bacterial communities of eight wild‐captured green turtles to four stranded turtles in the central Great Barrier Reef regions that include Bowen and Townsville. We used high‐throughput sequencing analysis targeting the hypervariable V1‐V3 regions of the bacterial 16S rRNA gene. At the phylum level, Firmicutes predominated among wild‐captured green turtles, followed by Bacteroidetes and Proteobacteria. In contrast, Proteobacteria (Gammaproteobacteria) was the most significantly dominant phylum among all stranded turtles, followed by Bacteroidetes and Firmicutes. In addition, Fusobacteria was also significantly abundant in stranded turtles. No significant differences were found between the wild‐captured turtles in Bowen and Townsville. At the family level, the core bacterial community consisted of 25 families that were identified in both the wild‐captured and stranded green turtles, while two unique sets of 14 families each were only found in stranded or wild‐captured turtles. The predominance of Bacteroides in all groups indicates the importance of these bacteria in turtle gut health. In terms of bacterial diversity and richness, wild‐captured green turtles showed a higher bacterial diversity and richness compared with stranded turtles. The marked differences in the bacterial communities between wild‐captured and stranded turtles suggest the possible dysbiosis in stranded turtles in addition to potential causal agents. &NA; Graphical Abstract Figure. Comparative analysis of the fecal bacterial communities of wild‐captured and stranded green turtles (Chelonia mydas).

Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the Haptophyta, Cryptophyta and SAR, before the photosynthetic Rhizaria acquired their green plastid. Additionally, plastidial ACCase was derived by HGT from an ancestor or relative of the Prasinophyceae and not by duplication of cytosolic ACCase.

Increased expression and secretion of recombinant hIFNγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris

Abstract Transcriptional co-regulation of adjacent genes has been observed for prokaryotic and eukaryotic organisms, alike. High levels of gene adjacency were also found in a wide variety of yeast species with a high frequency of co-regulated gene sets. The aim of this research was to study how selective pressure on the Histidinol dehydrogenase gene (HIS4), using amino acid starvation, affects the level of expression and secretion of the adjacent human interferon gamma gene (hIFNγ) in the recombinant Pichia pastoris GS115 strain, a histidine-deficient mutant. hIFNγ was cloned into the pPIC9 vector adjacent to the HIS4 gene, a gene essential for histidine biosynthesis, which was then transformed into P. pastoris. The transformed P. pastoris was cultured under continuous amino acid starvation in amino acid-free minimal medium for ten days, with five inoculations into unspent medium every second day. Under these conditions, only successfully transformed cells (hIFNγ -HIS4+) are able to synthesise histidine and therefore thrive. As shown by ELISA, amino acid starvation-induced selective pressure on HIS4 improved expression and secretion of the adjacent hIFNγ by 55% compared to unchallenged cells. RT-qPCR showed that there was also a positive correlation between duration of amino acid starvation and increased levels of the hIFNγ RNA transcripts. According to these results, it is suggested that these adjacent genes (hIFNγ and HIS4) in the transformed P. pastoris are transcriptionally co-regulated and their expression is synchronised. To the best of the knowledge of the authors; this is the first study demonstrating that amino acid starvationinduced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in P. pastoris.